key: cord-0810617-5ogip9tz
authors: Huang, Wanqiu; Lin, Dachuan; Wang, Cuini; Bao, Chaohui; Zhang, Zhaoqi; Chen, Xinchun; Zhang, Zheng; Huang, Jian
title: The determination of release from isolation of COVID-19 patients requires ultra-high sensitivity nucleic acid test technology
date: 2020-07-02
journal: J Infect
DOI: 10.1016/j.jinf.2020.06.075
sha: a7eb33ff9046852f17630a04082a86fe4ada8e87
doc_id: 810617
cord_uid: 5ogip9tz

nan

The prevention and control of SARS-CoV-2 has entered a critical period. Recent one paper in this journal also discussed weather qualitative RT-PCR be used to determine release from isolation of COVID-19 patients [1] . This issue is really important. Since the outbreak of COVID-19 worldwide, discontinuation of isolation has been presenting a dilemma of COVID-19, despite of the test-based strategy or the symptom-based strategy [1] . The reason for the confusion is that nucleic acid testing presents false negative based on qPCR technology, because of its low sensitivity [2] [3] [4] . There are several factors for false negative, including sample collection, preservation, transportation, virus inactivation, nucleic acid extraction and technical sensitivity, among which technical sensitivity and precise sampling are the most important quality control measures to eliminate false negative.

It is well known that SARS-CoV-2 nucleic acid test is the main diagnostic method of COVID-19. Recombinase polymerase amplification (RPA) is a new technology for testing nucleic acid with some advantages of simple operation, fast speed and low cost based on isothermal amplification. In our study, we developed an improved strategy, termed as nestRPA (nest recombinase polymerase amplification), which could greatly improve the sensitivity of nucleic acid detection for SARS-CoV-2 than RPA or qPCR. 4 Firstly, we designed eight sets of primers and probes for RPA on the conservation regions of SARS-CoV-2 genes, in which some fragments were designed to span multiple gene regions ( Figure 1A ) which is one of the important technical tips.

Through the two rounds of primer screening, we found that the limit of detection (LOD) of 16 pairs of primers and 8 probes is quite different ( Figure 1B ), in which Fragment 1 against ORF1 gene had the worst amplification efficiency. And Fragment 5 and 7 had the smallest LOD value, 300 and 500 copies/uL ( Figure 1C to 1F), respectively.

As far as we know, we firstly proposed the concept of nestRPA. The basic principles of nestRPA are as follows: in nestRPA, the first amplification fragment of target gene is amplified by outer primers, then a second fragment of target gene completely within the first amplification fragment is amplified by inner primers. In order to eliminate the influence of the fluorescence signal of enzymes, fluorescent probe is not included in first RPA reaction which is another important technical tips. And in the second RPA reaction, fluorescent probe will be added into reaction system. Using nestRPA technology, we found that positive plasmid containing SARS-CoV-2 with the concentration of 1 copy/ul could also be stably detected by Fragment 5 and nucleic acid detection results were negative using qPCR. However, we found 12.50% (4/32) of the samples were positive using Fragment 5 and/or Fragment 7 by nestRPA ( Figure 1I) , which was consistent with those reported by other researchers [5] . Our results suggested that the ultra-sensitive nucleic acid detection technique has important implications for early identification of those asymptomatic carriers infected with SARS-CoV-2. Of course, in order to avoid false positive results, the target sequence of positive amplification products was 100% detected by high-throughput sequencing. In summary, 36.18% (55/152) of the samples with qPCR negative results were identified as positive by nestRPA technology in 172 clinical samples from 127 patients, which indicated the analytical sensitivity of nestRPA assay is much better than that of qPCR ( Figure 1J ).

In addition, many experts of COVID-19 prevention and treatment clearly pointed out that the inaccurate sample collection were also one of the important reasons for the false negative result of SARS-CoV-2 nucleic acid [6] [7] [8] . The most commonly sites used as sampling are oropharynx and nasopharynx. The sample collectors should fix the tongue with a spatula, and the sampling swab is used to scrape the cells from tonsil recess and lateral wall when sampling from the oropharynx [9] . However, the sample collectors were often fear of contagion with SARS-CoV-2. Under great infection pressure, inaccurate sampling sites and inadequate sample volume will lead to false 7 negative test results. Therefore, it is helpful to reducing the false negative through strict and normative operation of precise sampling with well protection for sample collectors (Figure 2) .

Except for the technical sensitivity and precise sampling, we also need to pay more attention for the quality control of sample preservation and transportation, virus inactivation, nucleic acid extraction [10] . If all the links in the detection of SARS-CoV-2 nucleic acid could be strictly administrated, false negative could be completely eliminated, and the discontinuation of isolation will no longer be a dilemma for us.

All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Jian Huang was responsible for study concept and design. Zheng Zhang and Xinchun Chen were responsible for specimens sampling. Wanqiu Huang and Dachuan Lin were responsible for the experiment and statistical analysis. Wanqiu Huang, Dachuan Lin, Cuini Wang, Chaohui Bao and Zhaoqi Zhang were responsible for the analysis of data. Wanqiu Huang and Jian Huang were responsible for drafting the manuscript. 

Should qualitative RT-PCR be used to determine release from isolation of COVID-19 patients

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Negative Nasopharyngeal and Oropharyngeal Swabs Do Not Rule Out COVID-19

False-negative of RT-PCR and prolonged nucleic acid conversion in COVID-19:Rather than recurrence

Estimating the extent of asymptomatic COVID-19 and its potential for community transmission: systematic review and meta-analysis

Suboptimal biological sampling as a probable cause of false-negative COVID-19 diagnostic test results

Understanding the Influence Factors in Viral Nucleic Acid Test of 2019 Novel Coronavirus (2019-nCoV)

Expert consensus on specimen sampling technique of SARS-CoV-2 infected patients

Expert consensus on nucleic acid detection of COVID-19

Antibody Detection and Dynamic Characteristics in Patients with COVID-19